Actelion Pharmaceuticals Ltd., Allschwil, Switzerland.
PLoS One. 2012;7(10):e47662. doi: 10.1371/journal.pone.0047662. Epub 2012 Oct 15.
Two endothelin receptor antagonists (ERAs), bosentan and ambrisentan, are currently approved for the treatment of pulmonary arterial hypertension (PAH), a devastating disease involving an activated endothelin system and aberrant contraction and proliferation of pulmonary arterial smooth muscle cells (PASMC). The novel ERA macitentan has recently concluded testing in a Phase III morbidity/mortality clinical trial in PAH patients. Since the association and dissociation rates of G protein-coupled receptor antagonists can influence their pharmacological activity in vivo, we used human PASMC to characterize inhibitory potency and receptor inhibition kinetics of macitentan, ambrisentan and bosentan using calcium release and inositol-1-phosphate (IP(1)) assays. In calcium release assays macitentan, ambrisentan and bosentan were highly potent ERAs with K(b) values of 0.14 nM, 0.12 nM and 1.1 nM, respectively. Macitentan, but not ambrisentan and bosentan, displayed slow apparent receptor association kinetics as evidenced by increased antagonistic potency upon prolongation of antagonist pre-incubation times. In compound washout experiments, macitentan displayed a significantly lower receptor dissociation rate and longer receptor occupancy half-life (ROt(1/2)) compared to bosentan and ambrisentan (ROt(1/2):17 minutes versus 70 seconds and 40 seconds, respectively). Because of its lower dissociation rate macitentan behaved as an insurmountable antagonist in calcium release and IP(1) assays, and unlike bosentan and ambrisentan it blocked endothelin receptor activation across a wide range of endothelin-1 (ET-1) concentrations. However, prolongation of the ET-1 stimulation time beyond ROt(1/2) rendered macitentan a surmountable antagonist, revealing its competitive binding mode. Bosentan and ambrisentan behaved as surmountable antagonists irrespective of the assay duration and they lacked inhibitory activity at high ET-1 concentrations. Thus, macitentan is a competitive ERA with significantly slower receptor dissociation kinetics than the currently approved ERAs. Slow dissociation caused insurmountable antagonism in functional PASMC-based assays and this could contribute to an enhanced pharmacological activity of macitentan in ET-1-dependent pathologies.
两种内皮素受体拮抗剂（ERAs），波生坦和安立生坦，目前被批准用于治疗肺动脉高压（PAH），这是一种涉及激活的内皮素系统和肺动脉平滑肌细胞（PASMC）异常收缩和增殖的破坏性疾病。新型 ERA 马西替坦最近在 PAH 患者的 III 期发病率/死亡率临床试验中完成了测试。由于 G 蛋白偶联受体拮抗剂的结合和解离速率会影响其体内的药理学活性，我们使用人 PASMC 来表征马西替坦、安立生坦和波生坦对钙释放和肌醇-1-磷酸（IP（1））测定的抑制效力和受体抑制动力学。在钙释放测定中，马西替坦、安立生坦和波生坦均为高度有效的 ERA，K(b) 值分别为 0.14 nM、0.12 nM 和 1.1 nM。马西替坦，而不是安立生坦和波生坦，表现出缓慢的明显受体结合动力学，这表现为延长拮抗剂预孵育时间会增加拮抗效力。在化合物洗脱实验中，与波生坦和安立生坦相比，马西替坦显示出明显较低的受体解离率和更长的受体占有率半衰期（ROt(1/2)）（ROt(1/2)：17 分钟对 70 秒和 40 秒，分别）。由于其较低的解离率，马西替坦在钙释放和 IP（1）测定中表现为不可逾越的拮抗剂，与波生坦和安立生坦不同，它可阻断广泛范围内内皮素-1（ET-1）浓度的内皮素受体激活。然而，将 ET-1 刺激时间延长超过 ROt(1/2)会使马西替坦成为可逾越的拮抗剂，揭示其竞争性结合模式。无论测定时间如何，波生坦和安立生坦均表现为可逾越的拮抗剂，并且它们在高 ET-1 浓度下缺乏抑制活性。因此，马西替坦是一种具有明显较慢受体解离动力学的竞争性 ERA。在基于 PASMC 的功能性测定中，缓慢的解离导致不可逾越的拮抗作用，这可能有助于增强马西替坦在 ET-1 依赖性病理中的药理学活性。
